The objectives of the project are to uncover the mechanisms of gene control that turn on, and distinguish between, developmental programs of spore formation and matrix production by Bacillus subtilis. Spore formation has been traditionally viewed as a behavior of free-living cells, but we now understand that differentiation also occurs in the context of structured, multicellular communities (biofilms) consisting of chains of matrix-producing cells as well as spore-forming cells. Indeed, the mechanisms that govern entry into sporulation are intimately interwoven with those that govern matrix production. This proposal addresses important gaps in our understanding of sporulation and multicellularity with four specific aims: (1) We will identify the natural environmental signals that trigger spore formation and multicellularity by two sensor histidine kinases. (2) We will visualize cell fate switching between planktonic and matrix-producing states in real time using a newly devised microfluidic device. We will determine how a double-negative feedback loop controls switching and how cells discriminate between alternative fates of spore formation and matrix production. (3) We will determine how D-amino acids are produced late in the life cycle of the biofilm and how they trigger biofilm disassembly. We will determine the regulatory mechanisms that control the expression of the racemase genes that are responsible for D-amino acid production, how D-amino acids are incorporated into the peptidoglycan and how they trigger the release of an amyloid-fiber component of the matrix. (4) We will determine the full cascade of regulatory events that govern the differentiation of a cell into a spore, including the mechanisms that govern switching from one sigma factor to another. Understanding how D-amino acids cause biofilm disassembly will inform strategies for blocking biofilm formation by pathogenic bacteria. Also, research into gene control by B. subtilis, the principal model organism for Gram-positive bacteria, has provided, and will continue to provide, fundamental insights into the molecular biology of related, pathogenic bacteria, such as Staphylococcus, Enterococcus and B. anthracis.